Mechanism Of Platelet Aggregation Induced By Human Breast Cancer MCF-7 Cell Lines

Objetive:To investigate the molecular mechanisms implicated in human breast cancer MCF-7 cells induced platelet aggregation.Methods:1.Different concentration of MCF-7 cells (0, 0.05×106/ml, 0.5×106/ml, 1×106/ml, 5×106/ml, 50×106/ml)were used to induced platelet aggregation(TCIPA). The four inhibitors (aspirin, apyrase, 7E3, SZ-1), which acts on the different pathways of platelet activation, were used to intervene in MCF-7 induced TCIPA.2.Platelet aggregation was expressed as the maximal percentage of aggregation rate, which was detected by light aggregometry.3.The cell morphology during TCIPA was observed by phase-contrast microscopy.4.The fluorescence intensity of GPⅡb/Ⅲa and GPIb on the surface of platelets and tumour cells during TCIPA was measured by flow cytometry. The correlations between platelet aggregation rate and platelet membrane receptors GPⅡb/Ⅲa and GPⅠb at different time points during TCIPA were analysised.5.ELISA was used to detect the release of thromboxaneA2 during TCIPA.Result:1.The platelet maximal aggregation rates induced by MCF-7 cell at six different concentrations(0 ,0.05×106/ml,0.5×106/ml,1×106/ml,5×106/ml,50×106/ml)were 0;7.7±0.5%;21.7±1.2%;28.5±1.5%;43.3±1.7%;44.5±1.3%, respectively. The maximum platelet aggregation rate gradually increased with increasing of concentration of MCF-7. ( P<0.05 ) And the cell morphology of platelet aggregation was observed by phase-contrast microscopy.2.At 50% maximum platelet aggregation rate induced by MCF-7, the expression levels of GPⅡb/Ⅲa and GPⅠb receptors on platelets were (853±124, 137±21), and signicantly higher than these of control group(un-induced). The expression of GPⅡb/Ⅲa and GPⅠb receptors on MCF-7 cells were 178±22, 344±53. At different time points during TCIPA, platelet aggregation rate had positive correlation with expression of GPⅡb/Ⅲa and GPⅠb(r=0.968, P<0.05;r=0.998, P<0.05), the expression levels of GPⅡb/Ⅲa had positive correlation with expression of GPⅠb(r=0.971,P<0.05).The release level of TXB2 measured during TCIPA induced by MCF-7 cells had no significant change(79.5±7.9vs. 71.8±9.7)(P>0.05).3.Aspirin did not significantly inhibit the platelet aggregation rate induced by MCF-7(43.3±1.5% vs. 43.3±1.7%)(P>0.05). In contrast to aspirin, Apyrase,7E3,SZ-1 inhibited MCF-7-induced platelet aggregation rate significantly(21.5±1.3, 30.4±1.8, 26.7±1.2% vs. 43.3±1.7%)(P<0.05, P<0.05, P<0.05, respectively). Combined inhibitory effects of these compounds abolished TCIPA induced by MCF-7 cells significantly(P<0.05). Compared with all signal inhibitor groups, two inhibitor combination group had more significantly inhibitory effect(P<0.05).Compared with all two inhibitors combination groups, three inhibitor combination group had more significantly inhibitory effect(P<0.05). At the presence of inhibitors, the number and size of platelet clumps induced by MCF-7 were significantly reduced.4.Inhibitors of receptor glycoproteins could significantly inhibit expression of receptor glycoproteins on the surface of platelet(853±124% vs. 532±40%, 137±21% vs. 117±10%)(P<0.05, P<0.05, respectively)but not on the surface of MCF-7 cells(178±22% vs. 164±10%, 344±53% vs. 336±54)(P>0.05, P<0.05, respectively). Inhibitors of platelet activation had no effect on the release of TXB2 during TCIPA(P>0.05).Conclusions:1. Human breast cancer MCF-7 cells may induce platelet aggregation in a concentration-dependent manner.2. TCIPA significantly enhanced the expression levels of GPⅡb/Ⅲa and GPⅠb receptors on platelets. Inhibitors of ADP-, GPⅡb/Ⅲa-, GPⅠb-Ⅸ-mediated pathways of platelet activation can inhibit MCF-7 induced platelet aggregation. Joint use of these inhibitors can play a more significant inhibition on TCIPA induced by MCF-7 cells. These results indicate that ADP-, GPⅡb/Ⅲa-, GPⅠb-Ⅸ-mediated pathways of platelet activation may involve in MCF-7 cell induced platelet aggregation.3. In contrast, inhibitor of TXA2-mediated pathway dose not reduce MCF-7 induced platelet aggregation and the release levels of TXB2 during TCIPA induced by MCF-7 cells have no significant change. It suggested that TXA2-mediated pathway may not involve in MCF-7 cell induced platelet aggregation.